The proposed research is designed to define the limits and specificity of the cholesterol-digitonin reaction in tissues at the fine structural level. Plans are to localize free cholesterol in gallstones, gallbladders and livers of mice fed a stone-inducing diet, and to attempt to localize cholesterol during degeneration and regeneration of rat sciatic nerve myelin. Studies with similar goals will be done in human gallstones and gallbladders obtained at surgery. The problem of specificity of the digitonin reaction for electron microscopic autoradiography will be examined using ethanol-extracted tissues and gallbladder contents from mice with diet-induced gallstones, as well as with tissues from normal animals. In addition, artifacts observed in tissue fixed with glutaraldehyde-digitonin will be compared with in vitro mixtures of phospholipids, cholesterol, and proteins. In all cases, tritium-labeled digitonin will be used as a basis for electron microscopic autoradiography.